The present invention relates to a method of assaying biologically active substances using fine particles and a labelling agent used therefor.
Recently, methods for the quantitative determination of trace constituents according to antigen-antibody reactions have been employed in clinical laboratory examinations and also in research in the fields of medical science, veterinary science, pharmacy and microbiology.
In classical processes, biologically active substances have been assayed using an erythrocyte agglutination reaction or an immunodiffusion method. Now, there are being developed nephelometric methods wherein an immune complex formed by the antigen-antibody reaction is determined according to the light scattering function of particles suspended in a medium; immunoassay by labelling wherein an antibody or antigen is labelled with a fluorescent dye, a radioisotope or an enzyme and an antigen or antibody is measured; and a method wherein an antigen or antibody is fixed on fine particles of a synthetic polymer and degree of the aggregation of the fine particles caused by the presence of antibody or antigen is observed on a glass plate or microplate or the change in transmittance of light due to the aggregation is measured using a spectrophotometer.
Particularly, radioimmunoassay has been employed broadly as an analytical method having the highest sensitivity. However, this method has serious defects such as the danger of exposure or requiring special installation, since a radioisotope is used. Therefore, the development of an assay method with a high sensitivity to replace radioimmunoassay has been demanded. A method developed under these circumstances is enzyme immunoassay which can be operated easily and has a high sensitivity wherein an enzyme is used in place of the radioisotope and merits of the immunoassay by labelling are maintained. Enzyme immunoassay surely has a measurement sensitivity comparable to that of radioimmunoassay for some substances. However, the high sensitivity is not always exhibited in the measurement of all substances and reagents used in enzyme immunoassay are relatively expensive. Thus, enzyme immunoassay is still inferior to radioimmunoassay.
Generally, immunoassay methods such as radioimmunoassay may be classified into two methods with respect to their principles. One of them is the competitive method and the other is the sandwich technique. In the competitive method, into a sample liquid containing an antigen or antibody as a substance to be assayed is added the same substance of a known concentration which has been labelled with a labelling agent such as a radioisotope, then the corresponding antibody or antigen is mixed and reacted therewith to form an antigen-antibody complex. The complex and free substances which remain after the complex formation contain both labelled and unlabelled substances to be assayed. By measuring the amount of the labelled substance, a quantitative determination of the substances in the sample is effected. The other method, the sandwich technique comprises two steps. A solid phase on which a bonding partner capable of specifically bonding with a substance to be assayed has been fixed is prepared in advance, reacted with the specimen, and then separated from the liquid phase. In the next step, the substance to be assayed on the solid phase is reacted with a labelled bonding substance obtained by labelling a substance specifically reacting with the substance to be assayed using a radioisotope or the like, and the labelled substance in the solid phase or liquid phase is determined to assay the substance.
Thus, the sandwich technique comprises two steps and, in the first step, the substance to be assayed in the sample is specifically reacted with its bonding partner fixed on the solid phase, whereby only the substance to be assayed is bonded with the solid phase and other substances and ions are removed by washing. Therefore, substances other than the substance to be assayed in the sample, i.e., those interfering with the immunological reaction, are not introduced in the second step. Further, by this treatment, concentration of the substance to be assayed in the measurement liquid is also effected. This is advantageous from the viewpoint of measurement sensitivity and accuracy.
On the other hand, the operation of the competitive method is easier than that of the sandwich technique. According to the competitive method, even a substance having only one antigen determinant can be assayed, while in the sandwich technique, such a substance cannot be assayed.
An object of the present invention is to provide a safe, inexpensive, highly sensitive assay method which can be employed in place of the radioimmunoassay.
A further object of the invention is to provide a new, highly sensitive method of assaying a biologically active substance which comprises using fine particles both in the competitive method and in the sandwich technique.